None
The invention relates to the treatment of viral, bacterial, parasitic, benign and malignant proliferative diseases, neurodegenerative diseases, inflammatory diseases, immunological diseases, transplanted organ rejection diseases, and diseases produced by intoxication with heavy metals. The invention relates to the use of specific metal chelating agents including, furoic acid, thiophenecarboxylic acid and their derivatives, analogs and structurally related chemicals as pharmacological agents that can be use effectively to disrupt and inactivate specific transition metal ion containing zinc finger structural motifs in metalloproteins and enzymatically active transition metal ion containing sites in metalloproteinases, and other metal containing motifs structural or functional, which in turn, inactivate the pathogenic virus, pathogenic prokaryotic or eukaryotic cells which produces disease conditions. The invention also includes the inactivation of any newly created biological pathogens and their metalloprotein products heretofore not recognized, such as those use in bioterrorism Since radioactive and non-radioactive materials can intoxicate metalloenzyme systems involved in normal physiological functions, the agents of this invention are also intended to be used for decontamination of animals and patients exposed to heavy metals spontaneously or by the use in bioterrorism.
It will be appreciated that hereinafter the use of the following terms: 1) xe2x80x9cmetalloprotein disrupting agentxe2x80x9d encompasses all of the intended functions of the invention and method including antiviral, antiinfective, antiinflammatory, anticancer, and so on; 2) the broad term xe2x80x9cantiinfectivexe2x80x9d is intended to include antiviral, antibacterial, antifungal, antiparasitic activities, as well as actions against any other infective agent or organism whether natural or synthetic; 3) the term xe2x80x9cantiinflammatoryxe2x80x9d is intended to include any inflammatory response; and 4) the term xe2x80x9cdecontamination of heavy metalsxe2x80x9d is intended to include any use of these agents in the treatment of stable or radioactive heavy metal poisoning in man or animals. The term xe2x80x9cbiological response modifierxe2x80x9d is intended to encompass any change in the response of a prokaryotic or eukaryotic cell to a second agent after initial treatment with any of the agents of this invention. It will also be appreciated that the term xe2x80x9canti-inflammatoryxe2x80x9d is intended to include all inflammatory responses of a metazoan organism such as production of stress heat shock proteins, white blood cell infiltrates, swelling, pain, fever and so on. The term xe2x80x9canalgesicxe2x80x9d refers to a pain reliever agent that functions in inflammatory conditions.
The role of metal ion containing proteins in physiological actions and pathological responses including cancer, inflammation, proliferative diseases and infectious diseases have been intensively study by many researchers. The inventors have studied the important functions of proteins having amino acid sequences which specifically bind transition metal ions. For example, the inventors have determined the role of zinc finger proteins in cancer, proliferative diseases and viral diseases. Moreover, the inventors have determined the role of numerous metalloproteins, such as the role of iron-finger hormone receptor proteins and zinc finger ribosomal proteins in carcinogenesis and aging.
A review of the literature reveals the critical role of metal containing proteins in physiological actions and pathological responses including cancer, inflammation, proliferative, infectious diseases and heavy metal poisoning (Fernandez-Pol, J A, 2001). The inventors have studied the important function of proteins having amino acid sequences which bind metals, particularly transition metal ions. The inventors have determined the important role metalloproteins in proliferative, inflammatory and infectious diseases. In additions, the inventors have determined the role of other metal ion containing protein complexes, such as the role of iron finger proteins in aging and carcinogenesis.
From the evidence reviewed, one can infer that development of a variety of drugs that control or neutralize metalloproteins may lead to a new therapeutic approach directed at controlling and preventing a wide spectrum of viral diseases, bacterial diseases, fungal diseases, cancer and other diseases involving abnormal expression of metalloproteins. Furthermore, the results suggest that these agents may be useful to prevent transmission of viral diseases and prevent the progression of other diseases. This review of the literature not only points out the limits of our understanding of this system, but also indicates the need for the development of new agents to control metalloproteins.
The specific features, objectives, and advantages of the instant invention and its preferred embodiments will become apparent after review and comparison with the prior art as follows. Remarkably, the instant invention provides agents heretofore not recognized, that interact with high specificity with structural metalloproteins and metalloenzymes essential for viral and cellular functions. However, none of these chelators are specific for zinc, in fact, some of them are more specific for iron, and they may have chelated a variety of transition metals (29). Nevertheless, these studies indicate that zinc plays a complex role in a dose and time-dependent manner in apoptosis.
The recent targeting of individuals and groups with the anthrax bacterium (Bacillus anthracis) spores is of great concern, particularly because the pulmonary form of this disease is most often fatal. This indicates the need to develop new antibiotics that will rapidly and effectively destroy the B. anthracis which proliferates inside lung and lymph node macrophages. This invention contributes new wide-spectrum antibiotics suitable for the destruction of B. anthracis inside macrophages which cannot perform their bactericidal functions in the presence of such bacterium.
Although only a limited number of viruses are currently thought to be adequate for biowarfare, such as smallpox or Marburg virus, there is a large number of viruses that can be made suitable and highly dangerous through genetic engineering manipulation or other selection process. There are thousands of animal and human viruses that have the potential to serve that purpose. Those viral agents that have not been recognized previous as credible biowarfare agents pose a present and clear danger, because there is no protection in the form of vaccines for prevention, no credible therapies, and no detection. The potential for destructive pandemic consequences would be far greater than those of the anthrax bacterium and may be similar to that of ancient smallpox pandemics. An opportunity exist to urgently correct and counteract this situation by developing new effective, low toxicity wide-spectrum antiviral agents. This invention contributes new wide-spectrum anti-viral agents suitable for these purposes.
The next sections discuss the background of this invention with special reference to applications to abnormal physiology, infectious diseases, heavy metal toxicity, special applications to enzymes, and metal complexes (Table 1).
In this section the inventors summarize the experimental data on the role of a selected group of metalloproteins, particularly viral (v) and cellular (c) zinc finger proteins (ZFP) and iron containing proteins which are involved in cell proliferation, neovascularization, apoptosis, and viral infection. Furthermore, this review summarizes the data embracing the hypothesis that disruption of certain metalloproteins by novel pharmacological agents is a key factor in controlling viral and proliferative diseases. The primary goal of this review is to show the potential therapeutic applications of ZFP disrupting agents, zinc chelators and iron chelators in the control of viral diseases and cancer.
It is known that zinc or iron deficiency, resulting from exposure of culture cells to membrane-permeable Zn2+ or Fe2+-chelators, can induced apoptosis in virally transformed cells while normal cells remain unaffected under these conditions. Apoptosis is possibly due to simultaneous inactivation of vZFP, cZFP, and/or iron containing proteins, which are essential for maintenance of cellular and viral structure and which are activated in virally transformed cells. New insights concerning apoptosis, viral metalloproteins, and novel antiviral agents will also be reviewed.
From the evidence reviewed, one can infer that development of a variety of drugs that control or neutralize vZFP may lead to a new therapeutic approach directed at controlling and preventing a wide spectrum of viral diseases and cancer. Furthermore, the results suggest that these agents may be useful to prevent transmission of viral diseases. Finally, these data not only points out the limits of our understanding of these systems, but also directed the inventors to the creation of the novel agents of the instant invention.
Due to the central importance and essential functions of viral and cellular zinc-finger proteins, the literature on these topics is now rapidly expanding. Different aspects of ZFP functions, for example, in apoptosis induced by viruses, been reviewed in recent years. In this article we will concentrate on the available information about various zinc finger proteins of viruses, the drugs that inhibit their function, and cellular zinc finger proteins induced by virus infection in an attempt to critically evaluate some basic biological consequences of manipulating zinc finger proteins.
The background of the invention is presented as a brief summary of information with some initial hypothesis supported by experimental evidence at the molecular and clinical level. The inventors evaluate the role of a selected group of zinc finger proteins of cells and viruses involved in apoptosis and viral infection. Because of the importance of ribosomal proteins having zinc finger structures in viral replication and cancer, those functions also are reviewed. Moreover, the authors briefly address the role of heat shock zinc finger proteins. Finally, the inventors summarize the data that leads to the known information embracing the hypothesis that disruption of zinc finger proteins by novel antiviral and anticancer agents is a key factor in controlling viral diseases and cancer.
Apoptosis
Apoptosis, or single-cell death, is programmed cell death that occurs during normal homeostasis. Apoptosis is further defined by morphological and biochemical characteristics which are regulated by evolutionary conserved genetic pathways common to metazoan organisms. Apoptosis serves a complementary but opposite role to mitosis in normal homeostasis.
Apoptosis involves a complex network of pathways that interact with each other to be either pro-apoptotic or anti-apoptotic. Developmental or environmental stimuli activate or inactivate specific genes resulting in a series of biochemical degrading reactions that result in orderly cell disintegration. Apoptosis is induced by a variety of events such as viral infection, anticancer agents, radiation, growth factor deprivation, cytokines and hormones. Mutations of genes in this pathway eliminate the apoptotic response and have proven oncogenic. For example, over expressing the gene Bcl-2 or eliminating the gene p53 alters the susceptibility to apoptosis and allows the proliferation of damaged cells which frequently results in cancer.
Zinc Chelating Agents and Apoptosis
A recent review summarizes the evidence that apoptosis is modulated by intracellular excess or deficiency of Zn2+ and presents some mechanism by which Zn2+ may control apoptosis (Fernandez-Pol, et al, 2001). The major conclusions are: 1) zinc deficiency, resulting from dietary deprivation or exposure of cultured cells to membrane-permeable Zn2+ chelators induces apoptosis; 2) zinc supplementation with Zn2+ to the media of cell cultures, can prevent apoptosis; and 3) an intracellular pool of chelatable Zn+ plays a critical role in apoptosis, possibly by modulating the activity of endonucleases.
There is evidence that apoptosis is modulated by intracellular excess or deficiency of Zn2+. Fragmentation of DNA and cytolysis are inhibited in certain systems when Zn2+ (0.8 mM) is added to the culture medium, It is interesting to note that Ca2+/Mg2+-dependent endonuclease activity in isolated nuclei was inhibited when Zn2+ was added to the medium. These studies are consistent with the hypothesis that Zn2+ prevents apoptosis by blocking the activation or inhibiting the activity of Ca2+/Mg2+-dependent endonuclease. Numerous reports have shown that depletion of intracellular Zn2+ by chelation can trigger apoptosis in virally transformed cells. For example, when leukemia cells were exposed to 1,10-phenanthroline, a Zn2+/Fe2+ chelator, DNA fragmentation and cell death occurred, unless the chelator was neutralized by a transition metal ion added to the medium Similarly, picolinic acid (PA) a Zn2+/Fe2+ chelator, induces apoptosis in many cells, including leukemia cells by chelating a pool of intracellular Zn2+/Fe2+, since influx of Zn2+/Fe2+ prevented apoptosis in the presence of PA, while chelation of Zn2+/Fe2+ induced apoptosis.
Because Zn2+ plays a role in many cellular functions, and because it is an structural component of zinc finger proteins which are essential in cell replication, there are many sites in the apoptotic pathway that can be potentially modulate by zinc and zinc chelators. A number of investigators have shown that apoptosis can be induced if the intracellular level of Zn2+ are reduced using chelators. For example, N,N,Nxe2x80x2,Nxe2x80x2-tetrakis-2-pyridyl methyl-ethylene diamine (TPEN) added to cultured cells induces apoptosis. These experiments add additional support to the hypothesis that changes in intra- and extracellular zinc can modulate apoptosis. However, none of these chelators are specific for zinc, in fact, some of them are more specific for iron, and they may have chelated a variety of transition metals. Nevertheless, these studies indicate that zinc plays a complex role in a dose and time-dependent manner in apoptosis.
Viruses relevant to human disease such as Smallpox, Ebola virus, Marburg virus, Lassa virus, Papillomavirus, Herpesvirus, and Retroviruses, including the AIDS virus, are all capable of inducing apoptosis. Viruses encode genes that both stimulate and suppress apoptotic cell death. These viral proteins interact with cellular pro-apoptotic (death factors) and anti-apoptotic (survival factors). Viral (v) and cellular (c) Zinc finger proteins (ZFP) are involved in apoptotic cell death. A pool of chelatable intracellular Zn2+ plays a critical role in viral and cellular apoptosis, possibly by modulating ZFP structure. In virally transformed cells apoptosis can be induced by intracellular deficiency of Zn2+ while normal non-infected cells remain unaffected.
Since 1980, Fernandez-Pol et al are studying the modulation of both v-ZFP and c-ZFP by a class of novel Zn2+/Fe2+ chelating, broad-spectrum antiviral agents which may form ternary complexes with the zinc atoms contained in ZFP (42-60). In numerous experiments, we found that these wide-spectrum antiviral agents block viral replication and induced apoptosis in virally transformed cells in culture. These agents also interfere with abnormally expressed c-ZFP produced by spontaneously or radiation transformed cells in culture. Thus, these studies provide evidence for a close correlation between interference with ZFP of both viral and cellular origins and apoptosis in transformed but not in normal cells.
Iron and Zinc Finger Proteins
Transition metal ions at physiological concentrations, such as chromium, zinc, iron, cobalt, and copper, are essential elements for biological functions; however in higher quantities they are toxic (Fernandez-Pol, et al, 2001). Evidence indicates that elevated levels of iron contribute to carcinogenesis. Two main factors are important in iron induced oncogenesis: 1) The capacity of iron to generate highly reactive free radicals which damage DNA; and 2) the increase iron requirement by rapidly proliferating transformed cells, which is required for DNA replication (ribonucleotide reductase) and energy production (within the mitochondrial in key enzymes of the redox systems of the respiratory chain). Studies with iron chelating agents such as picolinic acid and desferoxamine have contributed significantly to the understanding of differential mechanisms of growth regulation in normal and transformed cells (Fernandez-Pol et al, 2001). It is known that iron induces mutagenesis and/or carcinogenesis, but the detail mechanism of iron-induced oncogenesis is unknown.
Initial in vitro studies have demonstrated the ability of cobalt and cadmium to structurally reconstitute the zinc finger domains in an active form. In contrast, nickel and copper bind to zinc finger proteins, but are unable to restore the DNA binding capacity. These studies suggest that heavy metal incorporation into zinc finger may be important in metal-induced toxicity. Recently, it has been found that an iron-substituted zinc finger may generate free radicals which damage DNA and potentially induced carcinogenesis. The estrogen receptor (ER) is a ligand-activated transcription factor whose DNA-binding domain (ERDBD) is of the type Cys4-Cys4, which coordinate two zinc atoms, forming two zinc finger domains. The capability of iron to replace zinc in zinc finger, denoted the iron finger, was demonstrated in a series of experiments both in vivo and in vitro. Iron has the ability to substitute for zinc in the ERDBD as demonstrated by mobility shift and methylation interference assays of iron finger, which show specific recognition of the estrogen response element. The DNA binding constants for both in vivo and in vitro iron-replaced zinc fingers were similar to that of the native zinc-containing finger. Atomic absorption analysis showed a ratio of 2:1 iron atoms/mol of ERDBD protein. Remarkably, the iron finger in the presence of hydrogen peroxide and ascorbate generates highly reactive free radicals (hydroxyl), producing a reproducible cleavage pattern to the DNA of the estrogen response element. The close proximity of the zinc finger to DNA, as found in the computer modeled structure, suggests that the iron-substituted zinc finger may generate free radicals while bound to genetic regulatory response elements, leading to degradation of DNA and/or carcinogenesis.
Zinc Finger Ribosomal Proteins
Fernandez-Pol et al have shown that human metallopanstimulin (MPS-1)/S27 ribosomal protein is a ubiquitous 9.4-kDa multifunctional xe2x80x9czinc fingerxe2x80x9d protein which is expressed at high levels in a wide variety of cultured proliferating cells and tumor tissues. The human MPS-1 gene and its relationship to human cancer cell growth was discovered by Fernandez-Pol et al in 1989, using human MDA-MB-468 breast cancer cells stimulated with specific growth factors and serum. Since that time, research has consistently demonstrated that both MPS-1 mRNA and protein are involved in cancer cell growth as demonstrated by increased levels of MPS-1 mRNA and protein found in numerous pathological tissue specimens obtained from various types of human cancers, such as prostate, breast, lung, colon, endometrium, uterine cervix, vulva, and melanoma. These results indicate that the MPS-1 antigen is a ubiquitous tumor marker which may be useful in detection and prognosis of various types of malignant neoplastic conditions. The results of other experiments indicate that MPS-1 is involved in protein synthesis, repair of damaged DNA, digestion of mutated mRNA, anti-apoptosis and rapid cell proliferation. Thus, the information available indicate that MPS-1 is a multifunctional S27 ribosomal protein relevant to numerous oncogenic processes which can be used as a ubiquitous tumor marker in various clinical assays. More recently, MPS-1/S27 ribosomal protein has been shown to be increased in virus infected cells, in parasites such as Toxoplasmosis and Malaria, in yeast proliferative capacity, and in macrophage activation in human melanomas NCBI, National Cancer Institute Data Bank; Fernandez-Pol, 2001).
It is important to note at this point that there are many reports indicating a connection between overexpression of some genes encoding ribosomal proteins and cancer. There is evidence that a number of other ribosomal proteins have additional functions separated from both the ribosome and protein synthesis. Zinc finger motifs are characteristics of numerous ribosomal proteins, allowing them to bind to nucleic acids. This binding ability offers a potential mechanism for ribosomal proteins to interfere in both transcriptional and translational mechanisms. For example, the rat ribosomal protein S3a is identical to the product of the rat Fte-1 gene which encodes the v-fos transformation effector. S3a is involved in the initiation of protein synthesis and is also related to proteins involved in the regulation of growth and the cell cycle. Rat ribosomal protein L10 is homologous to the Jun-binding protein and to a putative Wilm""s tumor suppressor. Taken together, the findings of ribosomal proteins with oncogenic, tumor supressor, or cell cycle functions, indicates extraribosomal functions of certain ribosomal proteins related to oncogenesis.
Zinc Finger Heat Shock Proteins and Viral Activation
The involvement of zinc fingers in protein-protein interactions extends beyond the control of gene expression. In numerous proteins the zinc finger domains have been implicated in mediating homodimerization or heterodimerization (Fernandez-Pol et al, 2001). Prokaryotes and eukaryotes express numerous heat shock proteins (Hsps) in response to stress, including heat shock, exposure to heavy metals, hormones and viral infections.
The stress response which include numerous forms of physiological and pathological stress is involved in viral infection. A prominent feature of this response is the synthesis of a discrete set of zinc finger proteins, known as the heat shock proteins, which at present are denoted molecular chaperons. During infection by certain viruses, heat shock proteins act as intracellular detectors that recognize malfolded proteins. Researchers have found that certain DNA viruses are able to activate heat shock proteins. For example, the Hsp70 (DnaK) is induced by adenovirus, herpes virus, cytomegalovirus, and other viruses.
One of the most interesting proteins involved in the viral infection response is the DnaJ, a heat shock protein which functions in the control of protein folding within the cell. DnaJ proteins contain two CCCC zinc finger motifs, defined by the J domain, which is essential for stimulation of the Hsp70 ATPase activity. Thus, the results indicate that there is a relationship between the stress response and the cytopathic effects of certain viruses such as herpes viruses, poxviruses, and hepatitis C viruses.
The response of cells to stress, such as exposure to UV radiation, chemicals, bacteria, parasites, fungus or viruses is also associated with the induction of heat shock proteins. Hsp70 has a protective role in inflammation, infection, and regulatory roles in cytokine biosynthesis. Hsp70 exists in the cells in equilibrium between its free state, in the cytoplasm, and its bound state, protecting proteins in the nucleolus, interacting with ribosomal proteins to either refold some of the unfolded ribosomal proteins or by solubilizing the denatured ribosomal proteins to facilitate their use and increase the turnover rate. During release as a result of the heat shock, and as the nucleolus begins to recover its normal activities, a significant proportion of Hsp70 returns to the cytoplasm. This protein-protein interaction may have important implications for viral replication.
Thus, cellular inflammatory responses to viral infection are part of the organism defense against viruses. Zinc finger proteins, therefore, may be a key to the control of the cellular inflammatory response. Agents which can modify the zinc finger heat shock proteins may be useful in controlling the stress response.
Viral Zinc Finger Proteins are Highly Conserved Structures
All viruses depend on their ability to infect cells and induce them to make more virus particles. If the virus is successful the cells almost invariably die in the process, and that process have been shown to be apoptosis in numerous instances. Other viruses can integrate its DNA in the cellular DNA and remain inactive for long periods. The nucleic acid genome of viruses is always surrounded by a protein shell, denoted capsid, which is composed of nucleocapsid proteins, and some viruses also have a lipid bilayer membrane, termed an envelope, which enclose the nucleocapsid proteins.
Viral ZFPs have been identified in at least two thirds of all viruses studied (Fernandez-Pol et al, 2001). Examples of families of viruses using metalloproteins such as ZFP, zinc ring proteins or transition metal ion-dependent enzymes for replication, packaging and virulence are Arenaviridae, Reoviridae, Rotaviridae, Retroviridae, Papillomavirinae, Influenza, Adenoviridae, Flaviviridae (Hepatitis C), Herpesviridae, and Orthomyxoviridae (Influenza viruses). Viral ZFP are structural virion proteins essential for viral replication and packaging of the virus inside infected cells. Deletion of zinc finger domains in specific vZFP is lethal to the virus. Since the zinc finger domains of vZFP are essential for viral survival functions, they are conserved throughout evolution and there are no known mutants of the vZFP domain(s). Because the viral zinc finger domain(s) represent indispensable site (s) on the vZFP that can be attacked by one or multiple drugs, vZFP are ideal and primary drug targets for the next generation of antiviral agents (Femandez-Pol et al, 2001).
A computer search of all known viruses reveals highly conserved structures in their nucleocapsid (NC) proteins and other essential viral proteins. All viral NC proteins contain sequences of about 20 amino acids with 4 invariant residues, CCHC or other combinations, which chelate zinc through histidine imidazole and cysteinic thiolates with a Kd les than 10xe2x88x9213. These structures are denoted viral zinc fingers, and are highly conserved in numerous families of viruses. Examples of viruses which posses zinc finger NC proteins and other zinc binding proteins are show in Table I. These metal binding proteins are highly conserved in nature, and they perform essential functions in viral infectivity. It has been shown that mutations of the chelating residues in the zinc fingers produces a non-infectious virus. Furthermore, chelating agents have been shown to inactivate viruses. Thus, disruption of these proteins by specific agents results in viral inactivation. It has been suggested that the course of numerous viral diseases, such as genital warts, genital herpes, smallpox, chickenpox, influenza, viral hepatitis, etc, can be altered by inhibiting essential viral metalloproteins utilized during the viral infection cycle (Fernandez-Pol et al, 2001).
Papilloma virus infection results in a number of proliferative diseases in humans including warts induced by type 4 human papilloma virus (common warts). Moreover, papilloma virus can cause plantar ulcers as well as plantar warts. Human papilloma virus infection of the uterine cervix is the most common of all sexually transmitted diseases. Commonly know as genital warts, this wide spread virus infection is a serious disease that potentially can develop into cervical cancer. Since the virus is permanently present in cells, infection recurs in a significant percentage of patients.
Condylomata acuninata, also denoted genital warts, are benign epithelial growths that occur in the genital and perianal areas and caused by a number of human papilloma viruses (HPV) including types 6, 11 and 54. These are low risk viruses which rarely progress to malignancy. However, high risk viruses such as HPV-16 and HPV-18 are associated with cervical intraepithelial cancer.
The actions of HPV are mediated by specific viral-encoded proteins which interact and/or modulate cellular DNA and proteins to produce abnormal growth and differentiation of cells. Two proteins of the HPV viral genome, E6 and E7, are well conserved among anogenital HPV""s and both contribute to the uncontrolled proliferation of basal cells characteristics of the lesions. The E7 oncoprotein is a multi-functional protein with transcriptional modulatory and cellular transforming properties. The E7 oncoprotein is a zinc finger protein.
A strong correlation between zinc binding and transactivation activity of E7 has been documented. The HPV-16 E6 protein is a zinc finger protein that binds DNA and has transcriptional activity which depends on the formation of the zinc fingers. E6 protein can complex with the cellular tumor suppressor protein p53 and it is necessary with E7 protein for the immortalization of primary human epithelial squamous cells. Only two proteins of HPV are consistently expressed and integrated in keratinocytes, the E6 and E7 zinc finger proteins. The E6 and E7 proteins are responsible for continuous cell proliferation. About twenty HPVs are associated with ano-genital lesions and all transformed keratinocytes of these lesions contain E6 and E7 zinc finger proteins. The E6 and E7 regulate growth and transformation by interfering with cellular p53 and pRb proteins, respectively. Thus, one should be able to control HPV by inactivating E6 and E7, the critical zinc finger proteins which are required for replication. When replication of the virus is halted, apoptosis of the virally-infected cells must occur. Thus, one can alter the epidemiology of, for example, carcinoma of the uterine cervix by interfering with the functions of zinc finger proteins.
Herpes viruses are highly disseminated in nature. Herpes viruses vary greatly in their biological properties and the clinical manifestations of diseases they cause. In humans eight herpes viruses have been isolated to date: 1) herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (H1SV-2), cytomegalovirus (HCMV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), human herpesvirus 6 and 7 (HHSV6 and HHSV-7). More recently the existence of HHV8 as a causative agent of Kaposi sarcoma has been documented. The known herpesviruses share two significant biological properties relevant to this invention: 1) all herpesviruses specify a large array of enzymes involved in nucleic acid metabolism, including ribonucleotide reductase, an iron containing enzyme; and 2) they possess major zinc finger DNA-binding proteins required for DNA replication.
Retrovirus virions contain a diploid genome consisting of an RNA complex formed by the association of two identical unspliced viral RNA molecules. In mature virions, RNA molecules are tightly bound to viral zinc finger proteins, denoted nucleocapsid proteins (Ncps). Retroviral Ncp is produced after the gag gene product (Pr55gag), has been processed by the viral protease. The Ncps are highly conserved in all known retroviruses. Point mutation of the cysteine and histidine residues of the zinc finger domain of NCp7 results in a radical reduction of genomic RNA packaging, and this results in a drastic decrease in viral infectivity. Further studies indicate that NCp7 plays a key role in several other steps of the viral life cycle.
The human immunodeficiency virus (HIV) encodes several regulatory proteins that are not present in other retroviruses. The nucleocapsid p7 protein of HIV has been targeted by the inventors and other investigators for treatment of UW viral infections. The p7 protein is required for the correct assembly of viral RNA in newly formed virus particles. The p7 protein contains two zinc fingers that are critical for the recognition and packaging of the viral RNA. Thus, agents that effectively attack the two zinc finger domains of the HIV virus nucleocapsid p7 protein inside infected cells will decrease the overall number of viral infective particles.
The influenza virus is not integrated with DNA and thus may be vulnerable to attack by the specific antiviral agents of this invention. The influenza viruses are dependent upon viral Zn2+ metalloproteases for specific viral functions. Processing of critical proteins of influenza virus is mediated by virus-encoded Z2+ metalloproteases. It is of interest for this invention that the most abundant virion protein and a type specific antigen of influenza viruses, the M1 protein, is a zinc finger protein. Furthermore, this protein is involved in packaging of the influenza virus. Thus, inhibition of influenza virus Zn2+ metalloproteinases and/or zinc finger protein M1 by the agents of this invention presents an opportunity for controlling the progression of influenza virus infection.
The poxviridae is a large family of complex DNA viruses that replicate in the cytoplasm of vertebrate and invertebrate cells. The most notorious virus of this family is the variola virus that causes smallpox. Infectious poxvirus particles contain a complex transcription system. A large number of virus-encoded enzymes and factors are packaged in the virus particle. For example, RNA polymerase, a zinc requiring enzyme, is involved in early transcription. Furthermore, both the small catalytic subunit and the large regulatory subunit of ribonucleotide reductase are virus-encoded proteins and closely resemble their eukaryotic counterparts both structurally (80% homology) and functionally. The synthesis of ribonucleotide reductase, is induced rapidly after vaccinia virus infection. Catalytic activity of the small subunit is inhibited by hydroxyurea. Furthermore, some of the early viral and cellular transcription factors utilized by the smallpox virus are zinc finger proteins.
Filoviruses, which cause deadly haemorragic fevers, are a large group of viruses that have non-segmented negative-strand (NNS) RNA as their genomes. The two main types are the Marburg and the Ebola virus. The nucleoproteins of these viruses interact with the linear RNA genome and also with cellular and ribosomal zinc finger proteins to perform specific viral functions. Thus, filoviruses are susceptible to inhibition by the agents of this invention.
There are numerous examples of families of viruses which utilize zinc finger proteins, zinc ring proteins and/or transition metal ion-dependent enzymes for specific viral functions. These viral proteins play an essential role in the structure, replication and/or virulence of viruses such as Reoviruses, Rotaviruses, Hepatitis C viruses as well as numerous other viruses.
Background
The National Cancer Institute has identified ZFP as the next target for antiviral drugs (USA Federal Register, 60, No. 154, 1995). Several laboratories are evaluating new antiviral drugs targeted to modify ZFP. These products are targeted towards modification of the amino acid cysteine, which is the binding site for zinc in zinc finger proteins. This section focuses on developments in the antiviral field related to inhibititors of zinc finger proteins of HIV, HPV, and HSV.
It has been known for many years that the structural and biological properties of viruses can be altered by chelating agents. For example, treatment of rotaviruses with chelating agents such as EDTA (10 mM) results in a single-shelled, double-layered, non-infectious viral particles. Moreover, in vitro exposure of various retroviruses to the chelating agents such as EDTA or EGTA in millimolar concentrations results in partial disintegration of viral membranes. Thus, disintegration and degradation of retroviruses and rotaviruses can be accomplished by chelating agents.
There are several chelating agents that eject the coordinately bound zinc atom from HIV zinc finger proteins. For example, Otzuka et al reported that novel zinc chelators inhibit the DNA-binding activity of zinc finger proteins of HIV. In addition, The Tat trans-activator, is a small protein of 75-130 amino acids, which may form a zinc-finger domain. Since HIV-1 lacking Tat replicates poorly and does not cause cytopathic effects, approaches to interfere with Tat may be useful in treating AIDS. The cysteine-rich domain of Tat binds divalent cations, either two Cd2+ or two Zn2+ atoms. Whether the cysteine-rich residues form a Zn2+ finger or a lattice binding pockets for divalent cations is unknown The pol gene also has a zinc finger amino acid sequence suggesting that chelation chemotherapy may have a role in the treatment of AIDS.
At least three efficient approaches may be used to design novel classes of inhibitors of viral ZFP activity that directly attack vZFP: 1) disruption of the zinc finger domain by modification of the cysteine residues which are the binding sites for Zn2+ in the vZFP, resulting in the ejection of zinc ion; 2) removal of the zinc from the zinc finger moiety by specific chelating agents, which results in inactivation of the vZFP; and 3) specific chelating agents that form a ternary complex at the site of zinc binding on vZFP, resulting in inhibition of the DNA or RNA binding activity of vZFP. Since these antiviral agents attack highly conserved structures in the virus they may circumvent the emergence of drug resistant mutants. Furthermore, the basic mechanisms of action of the novel antivirals (1 through 3, above) may be enhanced in viral disease if the antiviral agents which directly attack metalloproteins of the virus simultaneously attack cellular metalloproteins implicated in the pathogenesis of viral disease. Hence, the novel antivirals may also prove to be effective against cellular zinc finger-containing proteins such as ribosomal ZFP and heat shock proteins which are involved in viral infection. These cellular proteins are induced by the virus for specific viral functions such as replication, propagation, or as an inflammatory response of the cells to the virus.
The specificity of these agents may be due to cellular specificity, in which virally infected cells express cellular and viral ZFPs that are not expressed by normal uninfected cells in their basal or proliferative state. Another primary mode of action of these agents could be receptor specificity, in which vZFP act as receptors for specific zinc ejecting agents, or specific chelating agents which bind to vZFP and form an inactive ternary complex consisting of vZFP-Zn-chelating agent. Thus, vZFP may act as receptors for new agents that can form ternary complexes with vZFP.
Disruption of Viral Zinc Fingers of HIV by Chemical Agents
The HIV virus represents a daunting challenge for chemotherapy. A major problem with the treatment of HIV-1 infection is the emergence of drug resistance. HIV-1 nucleocapsid protein zinc fingers are targets of choice because they are mutationally intolerant and are required for both acute infection and virion assembly. After treatment with nontoxic disulfide-substituted benzamides which attack zinc fingers, the following has been observed with HIV virus: 1) inactivation of cell-free virions; 2) inhibition of acute and chronic viral infections; and 3) broad anti-retroviral activity. More important, resistant mutants have not been detected.
In all retroviruses, except spumaretroviruses, the nucleocapsid (NC) proteins contain one or two copies of a conserved zinc finger domain. This motif, represented as C-X2-C-X4-H-X4-C (C, cysteine; H, histidine; X, other amino acids), thereafter denoted a CCHC box or retroviral zinc finger, is different in different retroviruses, and coordinates a zinc ion in the virion. The histidine imidazole and cysteine thiolates chelate zinc (Kd less than 10xe2x88x9213). The CCHC box is one of the most highly conserved motifs in retroviruses. All mutations in the zinc finger domain which have been described to date have been lethal for the virus. The HIV-1 NC protein contains two zinc fingers separated by just 7 amino acids. The zinc fingers of NC p7 protein are required for both early events in virus infection and for packaging genomic RNA.
Rice et al proposed that the HIV-1 NC protein is an ideal target for drug development because of the following reasons: 1) the two zinc fingers are indispensable sites on the same protein that can be attack by a single drug; 2) Simultaneous mutations in each zinc finger would be required for the development of drug resistance; and 3) the close proximity of the targets greatly reduces the probability of recombinatorial events.
Numerous compounds that fracture the CCHC zinc finger also inactivate the HIV-1 virus. These products have been suggested to be effective for a broad range of human and animal diseases, including drugs to treat cancer and viral infections. The zinc fingers of retroviral NC proteins are prime antiviral targets because of the conservation of the Cys and His chelating residues and the absolute requirement of these fingers in both early and late phases of retroviral replication. Thus, drugs that interact with the CCHC zinc finger structures of HIV-1 and other retroviral nucleocapsid proteins may be extremely useful to halt the propagation of the virus.
A core HIV viral protein, denoted p7 nucleocapsid (NC), has two zinc fingers. The zinc fingers are involved in binding and packaging viral RNA into new virions which bud off from the infected cells. Experiments have demonstrated that when the two zinc fingers are deleted, the new budding virions are unable to incorporate RNA, resulting in non-infectious viral particles. Furthermore, the p7 NC protein participate in the process of reverse transcription. The exact role of p7 in this process is not clear, but it is thought that the p7 NC protein binds to the RNA molecule while the reverse transcriptase enzyme generates DNA from the RNA template. More important, HIV p7 NP in which the zinc fingers have been deleted is unable to infect susceptible cells. The zinc fingers are therefore essential for both phases of the viral life cycle: packaging and transcription.
A number of drugs that interact with zinc fingers work by ejecting Zn from the zinc finger, resulting in an a functional protein. The same drugs were active against HIV in tissue culture. One of the drugs is disulfiram (Antabuse), a drug that is frequently used for the treatment of alcoholism. However, this drug was ineffective in monkeys carrying SIV. Rice et al reported that C-nitroso compounds inactivate HIV-1 and eject zinc from the virus. Retroviral zinc-fingers of the type CCHC bind zinc stoichiometrically and with high affinity (dissociation constant Kd, about 10xe2x88x9212 M). Under physiological conditions, a 10-fold excess of EDTA removes only 50% of the zinc from the N-terminal CCHC zinc finger domain of the HIV-1 nucleocapsid protein, indicating that the chelating agent EDTA is inefficient in removing Zn from ZFP. To determine whether 3-nitrosobenzamide (NOBA) is capable of efficiently ejecting Zn from retroviral-type zinc-fingers, Rice et al used NMR to study an 18-residue peptide with sequence corresponding to the N-terminal zinc-finger domain of the HIV-1 nucleocapsid protein, denoted Zn (HIV-1-F1). The 1H-NMR spectrum showed that addition of NOBA results in the loss of signals generated by the zinc-bound histidine, and the appearance of zinc-free histidine signals.
Rice et al have also proposed that disulfide benzamides possess a wide-spectrum antiretroviral activity in cell culture by xe2x80x9cattackingxe2x80x9d the two zinc fingers of the HIV-1 nucleocapsid protein (NCp7) and ejecting the zinc. Nucleocapsid is present in the core of all retroviruses. NCp7 binds to the dimeric viral RNA genome. Mutagenesis of any of the cysteines of histidines in the ZF of HIV-1 NCp7 generates virions with defective RNA encapsidation and noninfectious particles. These observations indicate that compounds which specifically destroy the coordination of the Zinc to the NCp7 will have an antiviral effect. Further experiments demonstrated that the antiretroviral activity of these compounds is due to inactivation of NCp7.
Finally, one of the great problems with antiretroviral agents currently in use is the ability of the virus to generate mutants which are resistant to the therapeutic agent and which are able to replicate with the same efficiency as the wild-type. This problem could be solved if the target protein has no alternative structures capable of replacing the original target protein. The properties of the zinc fingers in numerous viral proteins indicate the they are the proper targets to avoid mutation: The zinc finger is absolutely conserved in retroviruses (except spumaretroviruses), and all mutants in the zinc finger are defective with respect to infectivity. However, it is unclear whether mutants defective in zinc fingers may arise in certain circumstances.
Inhibition of Viral and Cellular Ribonucleotide Reductases (RR) by Iron Chelating Agents: Implications for Therapy of HSV and HIV
Antivirals for the treatment of herpes infections such as acyclovir, ganciclovir and foscarnet have had a significant impact on the management of herpesvirus infections. However, the use of these agents has resulted in an increase emergence of drug-resistant virus strains. The need for new classes of anti-HSV compounds with novel mechanisms of viral inhibition is becoming increasingly apparent as mutants resistant to conventional antiviral agents emerge.
When a virus infects a cell, it could induce its host to make doxyribonucleotides for viral DNA replication by means of the cellular enzyme ribonucleotide reductase, or the virus, as in the case of HSV could carry its own specific RR genes which are expressed in the host cells and produce a new enzyme.
Iron chelators inactivate the RR of HSV. Since iron restores the activity of RR, the chelators inactivate the RR by directly removing its catalytically essential iron. Interestingly, there are certain chelators such as (348U87)2Fe and (A1110U)2Fe that also inactivate the viral RR. It is conceivable that the antiviral-Fe-RR forms a ternary complex that prevents the catalytic function of the Fe2+, labilazing the enzyme-bound iron to dissociation.
Numerous herpes viruses, such as herpes simplex (HIV-1 and HIV-2), Epstein-Barr virus (EBV), varicella-zoster virus (VZV), pseudorabies virus (PRV), and equine herpesvirus type I (EHV-1), and numerous other herpes viruses encode cellular ribonucleotide reductase (RR) activities. RR, which is formed by the association of two nonidentical subunits (R1 and R2), catalyzes the reduction of ribonucleoside diphosphates to their 2xe2x80x2-deoxy derivatives which are key intermediates in DNA biosynthesis. There is increasing evidence supporting the essentiality of RR in viral replication. Numerous organisms, including herpes viruses, bacteria, and mammals, encode ribonucleotide reductases the share a number of common characteristics. Two important characteristics of RR are the presence of a stable tyrosyl free radical and the dependency of Fe (III) for catalytic activity. The smaller (R2) subunit contains the iron and tyrosyl radical and the larger (R1) contains thiols which are redox active and provide the hydrogen for nucleotide reduction. The association of R1 and R2 are required for catalytic activity. Thus, a potential approach for antiviral therapy would be the utilization of peptides that can inhibit enzymatic activity by preventing the association of R1 and R2 subunits. However, since iron is required for catalytic activity a potential, less specific, strategy for antiviral therapy are iron chelating agents, which would deplete iron from the cells, and may have a significant activity against herpes viruses. In 1998 picolinic acid was tested at 3 to 1.5 mM on cultured Human Foreskin (HF) cells infected with HSV-2-strain G and it was found to cause apoptosis of HF infected cells. The specificity of the iron chelators may be cellular specificity rather than viral specificity: infected cells enter apoptosis versus non-infected cells which remain unaffected.
It is relevant to mention that cellular RR is not only an important virulence factor for herpes viruses, but that cellular RR is also involved in the virulence of HIV. It has been suggested that the inhibition of RR with agents such as hydroxyurea could have a possible application in the treatment of AIDS. Giacca et al have found synergistic antiviral actions of ribonucleotide reductase inhibitors and 3xe2x80x2-azido-3xe2x80x2-deoxythymidine on HIV-1. RR inhibitors reduce the cellular supply of DNA precursors (dNTP) by interfering with their de novo synthesis. A secondary effect is the stimulation of the uptake an phosphorylation of extracellular deoxynucleosides, including their analogs such as 3xe2x80x2-azidothymidine (AZT). Both effects are important to HIV replication, which requires dNTP and is impaired by the triphosphate of AZT. A clear synergism between AZT and RR inhibitors was observed at nontoxic doses.
The cancer phenotype consists of several distinct characteristics such as indefinite proliferative life span, anchorage-independent growth, low growth factor requirements, neovascularization, invasion and metastasis. A common characteristic of tumor cells is the constant overexpression of glycolytic and glutaminolytic enzymes, which results in altered carbohydrate metabolism In addition, cancerous cells can synthesize their own growth factors, which leads to cell proliferation that is independent of the otherwise carefully regulated supply of growth factors and growth-related hormones. Moreover, growth factors are instrumental in the invasive characteristics of cancer cells. For example, Vascular Endothelial Growth Factor (VEGF) activates the proliferation of endothelial cells which results in the creation of new blood vessels. Most interesting, growth factors can also activate matrix metalloproteinases (is) which are able to degrade the extracellular matrix. Remarkably, one of the prominent features of MMPs is that many of these genes are inducible by growth factors, cytokines, carcinogenic agents (e.g. phorbol esters), chemotherapeutic agents (actin stress fiber-disrupting drugs), radiation, and oncogenic cellular transformation. MMPs gene expression may also be down-regulated by transforming growth factors, retinoic acids and glucocorticoids. Thus, MMPs are fundamental enzymes in both the invasive process and metastatic disease and are susceptible to pharmacological control. The development of potent synthetic inhibitors of MMPs had led to clinical trials to treat patients with cancer.
There are growth factors and oncogenes produced by viruses which illustrate the complexity of the growth regulatory mechanism and the oncogenic process in vivo. For example, the complex smallpox virus which has recently acquire new notoriety due to its potential use in bioterrorism, contains a gene that encodes soluble EGF protein which promotes cell proliferation and is detected in all poxviruses genera. Furthermore, the family of poxviruses produce interleukin-l beta receptor which blocks IL-1 beta cellular defense activity. Tumor Necrosis Factor (TNF) is also produced by poxviruses and contributes to virulence in the form of apoptosis. All these poxviruses virokines and viroreceptors gene products contribute to the pathogenesis in the form of production of granulomatous proliferative lesions, and benign tumors as in the case of myxomatosis of rabbits. It has also been reported that the tat protein of the human immunodeficiency virus (TV), a viral regulatory gene product, possesses growth stimulatory activity in certain cell types. Oncogenic proteins of human papilloma viruses such as the E6 and E7 zinc finger proteins are also able to immortalize normal cells in vivo and in vitro. These oncogenic proteins are involved in human cancers such as the carcinoma of the uterine cervix.
The life spans of animals are genetically controlled and new data exist to support common mechanisms to control the number of times a cell will divide before it can no longer divide (senesce). A fundamental characteristic of normal cells is their limited ability to proliferate in culture. Invariably, after an initial mitotic period in culture, normal cells from humans and most other species suffer a gradual decline in their ability to proliferate. Eventually, the decline becomes irreversible. This progression towards a lower activity state has been termed xe2x80x9ccellular senescencexe2x80x9d. Cellular senescence has been studied most often in cultures of human fibroblasts (e.g. WI-38 cells). Numerous studies have indicated that cellular senescence in culture reflects aging in vivo. More recent studies have suggested that senescent fibroblasts are unable to proliferate, at least in part, because of selective repression of genes involved in transcriptional activity, such as a protooncogene designated as c-fos.
Cancer in humans and animals results from a multistep process which is described in experimental model systems as initiation, promotion, conversion, and progression. Each step in the process represents the selection of cells that have acquired the ability to surmount extra and intracellular growth regulatory signals. The cytogenetic evidence of multiple chromosome abnormalities in most tumor cells and the progressive aberrant chromosome structures that can be observed during tumor progression are also evidence for multistep process. Since the tumorigenic process is of rare occurrence, multiple levels of control must be operative to prevent the emergence of such cells in metazoan organisms.
The common cancers of the adult, including colon, lung, prostate and breast develop by stepwise accumulation of mutations affecting both oncogenes and tumor suppressor genes. These mutations accumulate gradually over time and extensive genetic changes are necessary to produce a highly malignant cell. For example, benign adenoma of the colon usually have a single gene change. As they progress toward malignancy they acquire 3 or 4 more gene alterations. These multiple changes may occur in a specific order. However, it is very likely that the number of changes rather than a precise sequence is important for cancer development. Thus, the non-specific cellular changes as the cell ages continue to contribute to shift the balance of control from tumor suppressor genes to oncogenes.
There are overlapping mechanisms that may be common to both cancer and aging. The loss of the efficient DNA repair-capacity is a major factor in both cancer progression and the aging process. One model for aging states that it is the result of accumulation of damage in the DNA genome with resulting loss of function of critical genes. It has been proposed that during the aging process, robust DNA repair events become less active or inefficient resulting in accumulation of damaged DNA, and eventually in death. One unifying concept simply states that failure to repair DNA damage in protooncogene or tumor suppressor genes causes loss of growth control and cancer. However if the accumulation of DNA damage does not involve these growth regulatory genes this simply leads to cell death or senescence.
The mechanisms by which numerous chelating agents work on specific metalloenzymes have been characterized. These mechanisms provide investigators with several metalloprotein targets. Specific drugs can be created that will inactivate the target metalloproteins. This patent application describes a group of novel chelating compounds that were designed for the specific control and inactivation of metalloproteinases.
Proteinases are enzymes whose function is the cleaving of protein chains at specific sites. They play a critical role in the physiology of viruses, prokaryotic and eukaryotic cells. Proteinases are essential for the processes of growth, would healing, tissue remodeling, immunological defense, digestion, apoptosis, and coagulation. Pathological activation or inactivation of these enzymes leads to numerous disorders that contribute to disease initiation and progression. These enzymes are targets for the development of proteinase inhibitors which result in numerous drugs for the treatment of diseases such as hypertension, coronary artery disease, asthma, inflammation, arthritis, cancer, metastasis, infectious diseases, cardiovascular, respiratory and neurodegenerative disorders.
Proteolytic enzymes are able to cleave peptide bonds and are characterized as hydrolases. Proteinases are further divided into four classes: serine proteases (I), cysteine proteases (II), aspartic proteases (II), and metalloproteinases (IV). The compounds of this invention refer exclusively to metalloproteinases and are proteinase inhibitors pharmacologically active at the metal ion containing active enzymatic site.
Modification of abnormal metalloproteinase activity represents an opportunity for controlling the initiation and progression of many diseases. The inventors and others have recognized that effective inhibitors of zinc containing metalloproteinases must have at least: 1) one functional group capable of binding to the catalytic zinc such as carboxylic acid, thiol, or hydroxamic acid; 2) have at least one functional group which can H-bond with the enzyme backbone; and 3) have one or more side chains capable of favorable London interactions with the enzyme active site. There are numerous examples of specific drugs that can be used to inhibit zinc metalloenzyme in accordance with the principles delineated above, for the purpose of controlling the initiation and progression of specific diseases. For example, a prime enzyme target in ocular hypertension is carbonic anhydrase (CA). CAs are metalloenzymes and many inhibitors of these enzymes are metal complexing anions that coordinate directly to zinc in the enzyme active site. Carbonic anhydrases are inhibited by sulfonamides that bind zinc and in the process substitute a catalytically important water molecule. Another example is the angiotensin-converting enzyme (ACE), a component of the renin-angiotensin system ACE is a zinc metalloenzyme that is inhibited by zinc chelators. For example, Captopril was designed to compete with angiotensin for the zinc ion in the enzyme and binds to zinc through a thiol group. Lisinopril, another ACE inhibitor binds to zinc through an amino carboxylate moiety. Based on these pharmacological concepts, the inventors present in this application examples of the successful or promising pharmaceutical applications of a novel type of metalloproteinase inhibitors.
In recent years major advances in elucidating the interaction between metalloproteins and therapeutic agents have allowed exact predictions for the drug binding sites. The exact nature of this interaction is critical to control drug specificity which in turn leads to the reduction of unwanted side effects. Structural information can be used to design molecules that bind to specific targets. The substrate specificity of enzymes allows the design of drugs with a well-defined specificity. Metalloenzymes are targets for inorganic drugs since metals play a key structural and catalytic role for numerous enzymes such as the zinc metalloenzymes that are one of the topics of this patent application. Neutralizing a metal that is essential to enzymatic action by another catytically incompetent metal can result in an inactive enzyme. This pharmacological action can be obtained by the coordination of an exogenous toxic metal to a specific chelating agent which will result in the substitution of the metal, or removal of the metal from the enzyme active site. The platinum drugs are one example in which the therapeutic effect of the drug is due to platinum while the chelating agent ligand is merely a carrier. Metal complexes of carbothioamides and thiosemicarbazones have also been found to inhibit ribonucleotide reductase and possess anticancer activity. Copper complexes and copper complexes of anti-inflammatory drugs have been shown to be effective as antiiflammatory agents. Furthermore, the matrix metalloproteinase matrilysin, a zinc enzyme, has been shown to be inactivated by cadmium, which forms an inactive Cd/Zn hybrid. It is evident from these works that complexes of metal ions can be very useful in many different disease conditions.
The coordination ability of metals allows the formation of strong attachments through covalent and ionic bonds. The term xe2x80x9cmetal complexesxe2x80x9d is interpreted to include complexes, compounds or ions. This compounds exert their therapeutic effect by binding to a metal site in an enzyme active site. The formation of the metal complex is central to their inhibitory actions.
Viruses utilize a discrete set of unique enzymes to perform their life cycles. Thus, these enzymes provide targets for antiviral drugs. Thiosemicarbazones have been used as antiviral agents. Thiosemicarbazones are also known to inhibit ribonucleotide reductase, RNA-dependent DNA polymerase, and dihydrofolate reductase. HIV-1 protease is an aspartyl protease that generates mature proteins from the products of the gag and pol genes. Many metal ions have been found to be inhibitors of HIV-1 protease. These observations together with the novel compounds of this invention may lead to the design of potent inhibitors of HIV-1 proteases which contain metals that can bind to the enzyme through ionic or covalent interactions and thus can be stronger inhibitors than the currently available.
Form the foregoing it appears that it would be beneficial to have a product that can interfere with the structure or action of certain zinc finger proteins or zinc metalloproteinases to stop the progression of certain infectious diseases, proliferative diseases, neurodegenerative diseases, and other diseases that depend upon zinc finger proteins and/or zinc metalloproteinases for the generation of the disease state. Furthermore, it would be beneficial to provide a product that can control these diseases by chelating metal ions from zinc-dependent, iron-dependent or copper-dependent proteins, enzymes, and/or hormone receptors necessary for the initiation, progression and maintenance of replication of cancer cells and other proliferative disease conditions. Likewise, it would be beneficial to inhibit angiogenesis in certain disease states, such as cancers, allograft rejection, retinopathies, and post-ophthalmic surgery.
It is evident that enzymes are natural targets for inorganic drugs since metals play a key enzymatic role for many enzymes, such as the zinc metalloproteinases. The coordinating ability of metals holds the attractive promise of forming stronger attachments. Thus, it would be beneficial to provide metal complexes that show effectiveness by providing inhibitory metal ions with the chelating agent merely as a carrier. Furthermore, since the products of this invention are chelating agents, they may be used to treat metal toxicity conditions such as iron, mercury or lead toxicity.
With the advent of detailed data banks and highly sophisticated molecular modeling, the design of organic-chelate-metal inhibitors has achieved a great level of precision in targeting viral enzymes. In addition, the advent of more detailed modeling techniques about enzyme structures has allowed rigorous characterization of drug-enzyme interactions. In this invention, we have sought specific targets which play key roles in cancer cell metabolism, which are unique to cancer cells or are differentially expressed in cancer cells. Likewise, we selected other metalloprotein targets which would be beneficial to stop the progression of other diseases such as inflammatory diseases.
It is evident from the published works that the role of metal ions in neurodegenerative diseases is complex. The novel agents of this invention can provide the penetrability in the CNS, the appropriate concentration range, and the right pharmacokinetic characteristics for prolong treatment which is required for these diseases. Treatment for these disorders tend to be long term rather than single dose, and the novel compounds of this invention can afford specific interactions with critical enzyme targets and metalloproteins involved in disease processes. This would separate regulatory processes form toxic effects of the pharmaceutical agents.
It is clear from the diversity of therapeutic applications of chelating agents described in the background of this invention that disease pathology correlates with abnormal metalloprotein and/or metalloenzyme activity and that both metalloprotein and metalloenzyme inhibition can be a powerful and versatile tool in the treatment of various diseases as it will be demonstrated later for the novel agents of this invention.
Based upon the foregoing, several classes of compounds have now been discovered which can be used to inactivate viruses, pathogenic prokaryotes and pathogenic eukaryotes cells. Moreover, the identification of selected target proteins in these organisms are also described. The compounds identified are either lead compounds for the development of drugs or candidates for antiviral, antifective, and other therapeutic uses. Not every compound showing reactivity with the target metalloprotein will be able to penetrate the virus or cells and attack the target metalloprotein, in addition some will be toxic. However, identifying specific chemical groups that can react with these proteins enables rational drug design.
This invention relates to the prophylactic and therapeutic treatment of a mammal hosting a pathogenic virus, prokaryotic pathogenic organism, or eukaryotic pathogenic cell by the systemic administration of compounds having the following formula 
Wherein R1, which can be singly or multiply substituted in any position of the thiophene ring not already substituted by R2, is selected from the group consisting of hydrogen, alkyl and substituted alkyl wherein the alkyl portion is from 1 to 21 carbon atoms, inclusive, and isomeric forms thereof, cycloalkyls and substituted cycloalkyl, substituted oxygen, substituted nitrogen, halogen, phenyl, and substituted phenyl, xe2x80x94(CH2)OHxe2x80x94, (CH2)xe2x80x94NR3R4, and isomeric forms thereof, wherein n is an integer of from 1 to 21, inclusive, R3 and R4 are H or alkyl of from 1 to 21 carbon atoms, inclusive, and isomeric forms thereof, wherein R2, which can be singly or multiply substituted in any position of the thiophene ring not already substituted by R1, is 
and X is the amino function of a compound selected from the group consisting of 2-hydrazine, 2-hydrazone, or 2-thiosemicarbazone; and the pharmaceutically acceptable acid-addition salts thereof, and to the use of compounds of the formula 
Wherein A, B, and D are selected from the group consisting of nitrogen, oxygen, sulfur, and CR1R2; R1 and R2 are as defined in claim 1, and can be attached to any ring carbon or nitrogen atom, the R1 can be multiply attached to any ring carbon atom; the R1 can be 2-carboxylic, 2-hydrazine, 2-hydrazone, and 2thiosemicarbazone; and the pharmaceutically acceptable acid-addition salts thereof.
Compounds of particular importance for the subject of this invention are the of the following formula: 
wherein R1 is in the 4 position and is a fatty acid of from 1 to 21 carbon atoms, inclusive, and isomeric forms thereof; wherein R2 is in the 3 or 5 position and is an halogen; and the pharmaceutically acceptable acid-addition salts thereof, to a mammal hosting a pathogenic organism or diseased cells.
Other compounds of particular importance for the subject invention, derived from the formula defined immediately above, have the following formulas:
4-bultyl-3-5-dichloro-2-thiophenecarboxylic acid hydrazine, 4-bultyl-3-5-dichloro-2-thiophenecarboxylic acid hydrazone and 4-bultyl-3-5-dichloro-2-thiophenecarboxylic acid thiosemicarbazone.
Furthermore, within the scope of the subject, invention are the use of compounds of the formula 
Wherein A, B, and D are selected from the group consisting of nitrogen, oxygen, sulfur, and CR1R2; R1 and R2 are as defined in claim 1, and can be attached to any ring carbon or nitrogen atom, the R1 can be multiply attached to any ring carbon atom; the R1 can be 2-carboxylic, 2-hydrazide, 2-hydrazone, and 2-thiosemicarbazone; and the pharmaceutically acceptable acid-addition salts thereof.
The chemistry and synthesis of one of the analogues described above can be described in exemplary form as shown in FIG. 2. This analogue have been shown to be 7-10 times more active than 2-thiophenecarboxylic acid against cancer cells in tissue culture.
More particularly, in one embodiment of the present invention, a method is provided for dissociating a zinc ion (or copper, or iron or other transition metal ion [TMI]) from zinc finger proteins or metalloproteinases, the method comprising contacting the metalloprotein with a compound selected from the group consisting of:
4-bultyl-3-5-dichloro-2-thiophenecarboxylic acid hydrazide, 4-bultyl-3-5-dichloro-2-thiophenecarboxylic acid hydrazone and 4-bultyl-3-5-dichloro-2-thiophenecarboxylic acid thiosemicarbazone or derivatives thereof.
A method for inactivating a metalloprotein or metalloenzyme of a pathogenic virus, pathogenic prokaryotic organisms or pathogenic eukaryotic cells, wherein the said protein comprises an amino acid sequence structure which chelates a zinc ion or a transition metal ion, said method comprising the step of contacting intravirally or intracellularly the said zinc ion or transition metal ion bound to the chelating protein structure, with a chelating compound which dissociates the metal ion protein complex selected from the group consisting of the following compounds:
Examples of such compounds include, but are not limited to the following:
1. Furoic acid having the formula described above;
2. 2-Thiophenecaboxylic acid having the formula shown in FIG. 1;
3. Halogenated furoic acid as defined in claim 1;
4. Halogenated 2-thiophenecarboxylic acid as defined in claim 1;
5. Hydrazides of furoic acid or 2-thiophenecarboxylic acid having the formula xe2x80x94Cxe2x80x94NHxe2x80x94NHxe2x80x94R, where xe2x80x94C is attached to position 2 of the thiophene ring;
6. Hydrazones of furoic acid or 2-thiophenecarboxylic acid having the formula xe2x80x94CHxe2x95x90Nxe2x80x94NHxe2x80x94Cxe2x95x90O, where xe2x80x94C is attached to position 2 of the thiophene ring;
7. Thiosemicarbazones having the formula xe2x80x94CHxe2x80x94Nxe2x80x94NHxe2x80x94Cxe2x95x90Sxe2x80x94, where C is in position 2 of the thiophene ring;
8. Halogenated furoic acid, halogenated 2-thiophenecarboxylic acid, and derivatives thereof where the halogen is selected from the group consisting of F, I, Br, and Cl;
The compounds of 1 to 8 in which one or more of the ring residues in positions 3 or 4 have been replaced by a fatty acid side chain of 2 to 21 carbons.
The compounds of 1 to 8 coordinately complexed to cupric ions, or to ferric ions, or to a toxic metal ion such as platinum.
A method for inactivating a metalloprotein or metalloenzyme of a pathogenic virus, pathogenic prokaryotic organisms or pathogenic eukaryotic cells, wherein the said protein comprises an amino acid sequence structure which chelates a zinc ion or a transition metal ion (TMI), said method comprising the step of contacting intravirally or intracellularly the said zinc ion or TMI bound to the chelating protein structure with a chelating compound which dissociates the metal ion from the protein complex, said chelating compound selected from the group consisting of the following compounds:
2-furoic acid; 2-furoic acid hydrazide; Tetrahydro-2-furoic acid; 3,5-dibromo-2-furoic acid;
3,4,5-tribromo-2-furoic acid; 2,5-dimethoxytetrahydro-2-furoic acid hydrazine;
5-[2-Chloro-5-(trifluoromethyl)phenyl]-2-furoic acid;
5-[3-(trifluoromethyl)phenyl]-2-furoic acid;
5-(2-Nitrophenyl)-2-furoic acid;
5-(3-Nitrophenyl)-2-furoic acid;
5-(4-Chloro-2-nitrophenyl)-2-furoic acid;
5-(4-Chlorophenyl)-2-furoic acid;
5-(4-Methyl-2-nitrophenyl)-2-furoic acid;
5-(4-Nitrophenyl)-2-furoic acid;
5-Bromo-2-furoic acid;
5-Chloro-2-furoic acid;
5-Nitro-2-furoic acid
5-Nitrofuran-2-carboxylic acid.
2-thiophenecarboxylic acid; 3,4,5-trichloro-2-thiophenecarboxylic acid; 2-thiophenecarboxylic acid hydrazide; 2-thiophenecarboxylic acid hydrazone;
3-methyl-2-thiophenecarboxylic acid;
5-bromo-2-thiophenecarboxylic acid hydrazone;
5-methyl-2-thiophenecarboxylic acid;
5-chloro-2-thiophenecarboxylic acid;
and 5-chloro-2-thiophenecarboxylic acid hydrazone.
In yet another embodiment of the present invention a method for selecting a compound capable of dissociating a zinc chelated with a CCCC [or a CHCH, or other permutations of C and H] zinc finger of a viral protein is as follows: a) contacting the CCCC zinc finger of the CCCC protein with a chelating agent; and b) detecting the dissociation of the zinc atom from the protein. Such chelating agents include:
2-furoic acid; 2-furoic acid hydrazide; Tetrahydro-2-furoic acid; 3,5-dibromo-2-furoic acid;
3,4,5-tribromo-2-furoic acid; 2,5-dimethoxytetrahydro-2-furoic acid hydrazine;
5-[2-Chloro-5-(trifluoromethyl)phenyl]-2-furoic acid;
5-[3-(trifluoromethyl)phenyl]-2-furoic acid;
5-(2-Nitrophenyl)-2-furoic acid;
5-(3-Nitrophenyl)-2-furoic acid;
5-(4-Chloro-2-nitrophenyl)-2-furoic acid;
5-(4-Chloro-phenyl)-2-furoic acid;
5-(4-Methyl-2-nitrophenyl)-2-furoic acid;
5-(4-Nitrophenyl)-2-furoic acid;
5-Bromo-2-furoic acid;
5-Chloro-2-furoic acid;
5-Nitro-2-furoic acid
5-Nitrofuran-2-carboxylic acid.
2-thiophenecarboxylic acid; 3,4,5-trichloro-2-thiophenecarboxylic acid; 2-thiophenecarboxylic acid hydrazide; 2-thiophenecarboxylic acid hydrazone;
3-methyl-2-thiophenecarboxylic acid;
5-bromo-2-thiophenecarboxylic acid hydrazone;
5-methyl-2-thiophenecarboxylic acid;
5-chloro-2-thiophenecarboxylic acid;
The target proteins include but are not limited to: 1) MPS-1/S27 ribosomal protein; 2) the viral proteins described in Table 2; 3) LF protein of the Anthrax bacteria, 4) amyloid beta monomeric precursors of Alzheimer""s disease; and 5) matrix metalloproteinases. The selection method utilizes detection techniques such as: Nuclear Magnetic Resonance (NMR), high pressure liquid chromatography (HPLC), capillary electrophoresis, immunoblotting, release of radioactive isotope of metal ion, detecting fluorescence, and detecting mobility changes in gel shift assays.
It is among the objects of the present invention to provide specifics compounds which can inhibit the growth and proliferation of cells by blocking the activity of metal ion-containing proteins and metalloproteases.
Another object of the present invention is to provide a compound which can retard the growth and proliferation of target infective organisms including viruses, bacteria, fungi parasites or other infective agents by blocking the activity of metal ion-containing proteins.
Another object of the present invention is to provide a compound which can retard the growth and proliferation of target viruses or virus infected cells by blocking the activity of transition metal ion-containing protein structures such as zinc finger proteins or metalloenzymes associated with viral replication.
Another object of the invention is to provide such a compound to retard angiogenesis in malignant tumors by inhibiting metalloenzymes associated with neovascularization.
It is still another object of the present invention to provide a compound that can retard the growth of premalignant and malignant cells such as virally, chemically and spontaneously transformed cells.
Another objects of the present invention is to provide a compound that can retard the growth of premalignant and malignant cells such as virally, chemically and spontaneously transformed cells and be administered by any acceptable route, including orally, with substantial effectiveness and minimal side effects.
It is also among the objects of the present invention to provide a new treatment for patients suffering from various forms of viral infections by utilizing the novel properties of metal chelating agents as a chemotherapeutic anti-viral agent.
It is another object of the present invention to provide an agent that can halt the proliferation and transmission of viruses containing zinc finger proteins or metalloproteinases as essential viral proteins.
Another object of the invention is to provide a method of disrupting the function of metal containing protein structures containing metals other than zinc, such as iron-finger and other proteins with metal binding motifs heretofore unidentified by the administration of a metal chelating agent, both topically and systemically
Another object of the invention is to provide a product which can be spray in the nostrils or inhaled to prevent or control upper respiratory diseases such as influenza, rhinoviruses or pulmonary cancer.
Another object of the invention is to provide an anti-inflammatory compound that is effective in a broad range of inflammatory disorders including inflammatory response to infections and to chemical damage or radiation including, but not limited to, ultraviolet, atomic or medical radiation.
Yet another object of the invention is to provide such chelating agents in a relatively safe and nontoxic form such as 2-thiophenecarboxylic acid, its derivatives or related or similar compounds for both topical and systemic use.
Another object of the invention is to provide a topical preparation of metal chelating agents such as furoic acid, 2-thiophenecarboxylic acid (2-TH) or its derivatives to treat virally induced or spontaneous proliferative diseases of the skin or mucous membranes in human and animal subjects.
It is still another object of the present invention to provide an intravaginal preparation containing metal chelating agents such as furoic acid, 2-TH acid, or derivatives thereof that can prevent or retard sexually transmitted diseases caused by viruses or other causative agents containing zinc finger proteins or other zinc binding motif in their structure.
Still another object of the present invention is to provide a preparation containing chelating agents such as furoic acid, 2-TH acid or derivatives thereof that halts the progression of viral infections or proliferative diseases that is non-toxic to normal cells, relatively inexpensive and well suited for its intended purposes.
According to the invention, the main object is to provide a method of treatment and compound used in the method, for example, metal chelating compounds, such as 2-thiophenecarboxylic acid or derivatives thereof, for the treatment of infective or proliferative diseases, inflammatory responses, and cancers in human and animal subjects. The invention can be used orally or topically to treat or control a wide spectrum of proliferative diseases or conditions, both spontaneous or induced by viruses, bacteria, fungi parasites, chemicals, or radiation. The metal chelating compounds bind metal, for example iron or transition metal ions such as zinc, required by enzymes, or by transcription proteins found in viruses or malignant cells. By way of further example, the metal chelating compound, for example 2-thiophenecarboxylic acid or its derivatives, is used to bind the zinc contained in the zinc finger protein M1 common to the influenza viruses strains, thereby inactivating the virus and preventing the exit of RNA containing viruses or particles from the cells.
In one embodiment of the invention 2-TH acid in 500 mg capsules given in dosages ranging from 500 mg per day to 2000 mg per day, or more has been demonstrated by mathematical modeling to be effective in reducing the size of tumors, such as cancerous lymph nodes and inducing apoptosis in the cancerous tumor cells.
One embodiment of a topical preparation consists of a solution of the chelator, for example, 0.01% to 99%, preferably 5% to 25%, furoic acid or 2-TH acid in an appropriate vehicle, such as deionized water, buffer or other solvent, and is applied to the lesion three times a day. The preparation can be applied to skin to control acne, warts, herpes infections and to toe nails, to treat fungal infections. In another embodiment, the topical preparation consists of an ointment or cream containing approximately 0.5% to 99%, preferably 5% to 10% of furoic acid or 2-TH acid which is applied once or twice daily to the lesion. The ointment or cream can be instilled intravaginally to retard sexually transmitted viral diseases.
The various embodiments of the topical preparations can be used to treat papilloma and herpes viral diseases and to retard the papilloma, herpes and HIV viruses as well as proliferative diseases such as psoriasis and skin cancer.
Various derivatives that maintain their activity and stability after systemic administration are provided. Slow release oral formulations can be used to treat diseases for the digestive tract. The active derivatives can be administered orally, parenterally, by inhalation, transdermally or by any other appropriate method to control proliferative diseases, cancers, viral infections, HIV, and pulmonary Anthrax or any other condition wherein the causative agent includes a zinc-containing protein, whether the zinc-containing protein is a zinc finger protein, a zinc ring protein, or other type of zinc or metal containing structure heretofore unidentified or undetected, wherein the metal containing segment is required for protein stability and configuration and/or enzymatic activity.
It will be appreciated that other appropriate chelating materials such as the derivative of 2-TH acid may be used. It also will be appreciated that, although 1% to 5% topical preparations of furoic acid and 2-TH acid are described, a broader range of concentrations may be used. Further, the systemic doses may be altered or adjusted to ranges greater or lesser than those described, depending on toxicity and patient response, without departing from the scope of the appended claims.
It will be appreciated that some of the hydrophobic compounds of this invention can act intracellularly at low concentrations (pM to uM). Thus, the agents of this invention can penetrate cells, reach the target metalloprotein which is present at low intracellular concentrations (uM) and inactivate it.
It will be also appreciated that while the dose-response relationships of the chelating drugs of the prior art in general have a sharp square wave for the dose-response relationship and are non-specific, the compounds of the instant invention are target specific and have dose-response relationships for systemic use that are sigmoidal and thus have a wide range of therapeutic concentrations. Therefore, the agents of the instant invention are highly compatible with acceptable target to background therapeutic/toxic ratios.
Other features, objectives and advantages of the invention and its preferred embodiments will become apparent from the detail description which follows. Table 1 summarizes some of the medical applications of the agents of this invention.